Coherence microscope using interference of time incoherent light to achieve depth resolution in a measurement specimen

ABSTRACT

A coherence microscope has a divider ( 3 ) that divides light emitted by a light source ( 1 ) into measurement light, which is supplied to and reflected by a specimen ( 13 ), and reference light. A superimposition device ( 25, 31 ) superimposes the measurement light reflected by the specimen ( 13 ) with the reference light. A short sensor array ( 41 ) detects the light resulting from the superimposition and permits a read-out rate of at least about 60 kHz. The superimposition device has an emission device ( 25, 31 ) for emitting the measurement light and the reference light arranged to effect extensive irradiation of the sensor array ( 41 ) with superimposed light. The ratio of distances covered by the measurement light and the reference light from the emission device ( 25, 31 ) to impingement points on the sensor array ( 41 ) varies in the portion of the sensor array ( 41 ) that is irradiated with superimposed light.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention concerns a coherence microscope and a method of operatingsuch a microscope.

2. Description of the Related Art

In conventional microscopy problems are involved in the sharprepresentation of spatially extensive objects. Image sharpness isadversely affected due to blurred contributions of object regions aboveand below the focal plane. Different apparatuses and methods of imagingspatially extensive objects have therefore been developed.

One method with which sharp images can be obtained from spatiallyextensive objects utilises a confocal optical system for the imagingprocedure. The concept of imaging by means of a confocal optical systemis described for example in U.S. Pat. No. 3,013,467. That concept isbased on the fact that a light source in point form is made availablefor example by means of an aperture member with a small hole (pinhole),the light of that light source being focussed on to a point on thespecimen. The light reflected by that point on the specimen is in turnfocussed on to a point which represents an image of the point of thespecimen. Arranged at the location of that image is a second pinhole,behind which is disposed a detector for detecting the reflected light.Only light originating from the focal plane is projected on to a pointat the location of the second aperture member and can pass through thepinhole. Light which has been reflected at the specimen in front of orbehind the focal plane in contrast forms a disk at the location of thesecond aperture member. Such light therefore cannot pass through thepinhole so that essentially only light from the focal point reaches thedetector. Accordingly images of even spatially extensive objects can besharply produced by a confocal imaging procedure as contributions fromregions of the object which are above or below the focal plane are notinvolved in the imaging procedure. Slot apertures can also be usedinstead of pinholes. In that case the image of the light source on thespecimen is in the form of a line and the image of the light reflectedby the specimen appears as a line on the same or a further slotaperture.

Confocal microscopes, that is to say microscopes based on confocalimaging, are used for example as laser scan microscopes (LSM) inparticular in biology, material science and medical diagnostics. Interms of intraoperative diagnostics the particular challenge on thecorresponding microscopic scanning method is to be fast, of highresolution and compatible for use in an endoscope. Methods of that kindcan be used to effect optical biopsies for tumor detection for examplein the gastrointestinal tract.

A confocal laser scan microscope in conjunction with an endoscope is forexample the laser scan microendoscope described in Y S Sabharwal et al‘Slit Scanning Confocal Microendoscope for High Resolution In-VivoImaging’, Appl Opt 34, pages 7133-7144 (1999). That instrument producesthe image of a laser beam by means of a confocal aperture member on thespecimen, scans the specimen with the laser beam two-dimensionally(laterally) and receives the stray light reflected by the specimen. Inorder to record a spatial, that is to say three-dimensional image,two-dimensional planes are scanned at various depths. The depth of theplane to be recorded is adjusted by displacement of the focal plane ofthe microscope in the specimen. The result of that method is a so-calledz-stack of two-dimensional images. The scans are either implementedmanually (with a high degree of inaccuracy and a lack ofreproducibility) or by means of miniaturised focusing devices which mustsatisfy high demands in terms of accuracy and reproducibility and whichin addition are to be only of a small size. The mechanical demands onsuch focusing devices are very high. Laser scan microendoscopes havetherefore not yet become commercial products.

Longitudinal resolution of the laser scan microendoscope is determinedby the confocal optical system. Confocal imaging by means of an aperturemember provides that only stray light from a longitudinally closelydelimited region in respect of depth impinges on the detector. Thedepthwise extent of that region and thus the longitudinal resolution ofthe microscope depends on the opening in the aperture member, that is tosay for example the hole of the aperture diaphragm, and in practicereaches values of typically less than 10 μm. Better levels oflongitudinal resolution, that is to say more narrowly delimiteddepthwise regions, are possible by further closure of the apertureopening, which however is linked to a high degree of light loss.Disadvantages of the laser scan microendoscope are the long scanningtime which is necessary to record a z-stack and a low level of opticalsensitivity. Optical sensitivity is adversely affected by the generallylow degree of transmission of the optical fiber bundle of the laser scanmicroendoscope and troublesome reflections at the optical surfaces.

An alternative for imaging spatially extensive objects which is notbased on the principle of confocal imaging is the apparatus described inDE 199 29 406 for performing optical coherence tomography (OCT). Itincludes a light source which emits substantially incoherent light and abeam producing device for producing a measurement light beam and areference light beam which is coherent with respect to a reference timerelative to the measurement light beam, from the incoherent light. Thespecimen is irradiated with the measurement light beam. The lightreflected by the specimen is picked up and spatially superimposed withthe reference light beam. Because of the incoherence in respect of timeof the radiation, interference phenomena occur in the superimpositionoperation only with substantially identical optical travel lengths ofthe measurement light beam and the reference light beam. Thereforedifferent optical travel lengths for the reference light beam result ininterference phenomena with measurement light reflected by the object atdifferent depths. Thus the different optical travel lengths of thereference light beam can be related to the depth at which reflection ofthe measurement light took place in the specimen in order to obtain adepth profile of the specimen.

A disadvantage of the apparatuses set forth is that they cannot be usedto implement an optical biopsy in the desired manner.

Therefore the object of the invention is to provide an apparatus withwhich an optical biopsy can be implemented in a manner which isadvantageous in comparison with the state of the art. A further objectof the invention is to provide a method of operating such an apparatus.

SUMMARY OF THE INVENTION

According to the invention a coherence microscope includes a lightsource emitting light which is incoherent in respect of time. In thatrespect any light source of suitably short coherence length is deemed tobe a light source which is incoherent in respect of time. In additionthe coherence microscope includes a divider for dividing the lightemitted by the light source into measurement light which is supplied toa specimen and reflected thereby, and reference light. The apparatusalso has a superimposition device for spatial superimposition of themeasurement light reflected by the specimen with the reference light, aswell as a sensor line for detection of the light resulting from thesuperimposition, which is so adapted that it permits a read-out rate ofat least about 60 kHz. In order to achieve such read-out rates, it ispossible to use in particular short sensor lines with at most about 1000sensor elements, for example CCD elements (CCD: charge coupled device)and in particular very short sensor lines with at most about 500 sensorelements. The superimposition device has an emission device for emittingthe measurement light and the reference light, which is so adapted andarranged relative to the sensor line that extensive irradiation at leastof a part of the sensor line with superimposed light is effected and theratio of the distances covered by the measurement light and thereference light from the emission device to the respective impingementpoint on the sensor line varies in the portion of the sensor line thatis irradiated with superimposed light.

The coherence microprocessor according to the invention is based on thefollowing considerations:

A main obstacle in terms of effecting an optical biopsy with thedescribed state of the art is that the recording times for recording az-stack are long.

In the LSM, the cause of this is inter alia that, for recording az-stack, the specimen has to be raster-scanned a plurality of times insuccession at different depths, in which case the focal plane of themicroscope optical system has to be re-set in each case for varying thedepth. The setting operation requires a mechanical movement of opticalelements, which cannot be effected at the desired speed. Added to thisis the fact that both a high degree of lateral resolution and also ahigh degree of longitudinal resolution are desired for an opticalbiopsy. A high degree of longitudinal resolution however requires areduction in the aperture opening, which results in high light losses.

Just as in the case of the LSM, mechanical movement is also necessary ina standard OCT in order to arrive at items of image information fromvarious depths in the specimen. In apparatuses of that kind, depthwisedetermination is effected on the basis of the interference of ameasurement beam with a reference beam. In that case, the depth fromwhich the image information comes is ascertained from the distancecovered by the reference beam to the detector. That distance is usuallyvaried by the reference beam being reflected at a displaceable mirror.Therefore, to alter the depth from which the image informationoriginates, the mirror position must be mechanically altered, which,like the movement of the optical elements in the LSM, cannot be effectedat the desired speed.

The so-called line OCT described in DE 199 29 406, unlike a standardOCT, does not require a displaceable mirror in order to ascertain thespecimen depth from which the image information originates. In thatapparatus the light from the superimposition device is irradiated on tothe sensor line in such a way that the distance covered by the referencelight beam depends on the impingement point of the light on the sensorline. In that apparatus therefore the specimen depth arises out of theposition of the impingement point of the superimposed light on thesensor line, that is to say the position of the sensor element which isrespectively read out. Mechanical displacement of the mirror istherefore eliminated.

If in a line OCT the sensor line is selected in such a way that read-outof the sensor line can take place at a high read-out rate, that is tosay about 60 kHz or more, then the short recording times necessary forthe optical biopsy can be achieved. At the present time sensor linesenjoying the desired read-out rates and a line length of between 128 and1024 sensor elements are commercially available. The sensor line istherefore preferably a short line which includes not more than about1000 sensor elements. Particularly if a very high read-out rate is to beachieved, a very short sensor line with not more than about 500 sensorelements is preferably used.

A short or very short sensor line however does not necessarily have tobe used. Instead, it is also possible to employ a long sensor line, thatis to say a sensor line with more than about 1000 sensor elements, forexample a line with 2048 or 4096 sensor elements. In that case the highread-out rate can be achieved by only a respective portion of the sensorelements being irradiated and read out, that is to say the length of theline which is used is less than the actual length of the line. If forexample a sensor line with 2048 sensor elements is used, then preferablyonly about 1000 sensor elements of the line are irradiated and read out.Further preferably only about 500 sensor elements are irradiated andread out.

When reference is made to the length of a sensor line in the descriptionor the claims, that is intended to mean not exclusively the actuallength of the line but also the used length of a sensor line whoseactual length exceeds the length used.

The short sensor line and the elimination of moved parts for carryingout a depth scan permit the short recording times necessary for anoptical biopsy.

In addition to being distinguished by the short recording times thecoherence microscope of the invention is also distinguished inparticular by the following points, in comparison with the state of theart:

1) In comparison with a laser scan microscope (LSM) the coherencemicroscope is distinguished by a substantially higher level of signalsensitivity as the detection principle, as in the case of the OCT, isbased on the measurement of amplitudes and not on the measurement ofintensities. The dynamic range of detection is therefore greater byseveral orders of magnitude than in a conventional light microscope.That fact is of advantage in particular in terms of use in confocalfiber microscopy which operates with a low signal level.

2) The higher level of signal sensitivity of the coherence microscopehas the qualitative advantage over a fiber LSM, in terms of use inconfocal fiber microscopy, that both regions of high transparency andalso regions of high optical density can be better detected. Thatproperty is of particular interest in terms of optical biopsies.

3) In contrast both to the conventional mode of operation of a laserscan microscope and also that of a conventional OCT, a completelongitudinal scan (z-scan) is statically performed with the coherencemicroscope according to the invention at any point in the lateralspecimen plane (XY-plane) at high speed. The recording of z-stacks istherefore not necessary with specimen depths of typically 100 μm.

4) The advantage of static recording of a complete z-scan, referred toin point 3, permits a simplification in the scanning procedure. Insteadof a complete XY-scan (a so-called A-scan), the specimen can also berastered along a XZ-plane, that is to say only scanned along an X-line(a so-called B-scan), in which respect the X-direction is adjustable inits orientation and its ‘width’ without new positioning of themicroscope optical system which is possibly integrated into an endoscopebeing necessary. That permits a very fast optical biopsy which providesthe pathologist with a section in the accustomed orientation. The widthof the one-dimensional line can be adapted in particular to the desiredresolution and/or the desired signal strength.

The depth region which is accessible for measurement with the opticalcoherence microscope according to the invention by superimposition ofmeasurement and reference light, that is to say the depth extent of thedepth profile resulting from the superimposition, is referred to as thedepth variation. The depth variation is independent of depth resolutionand is determined by the number of sensor elements in the sensor line,the wavelength of the light used and the number of sensor elements perperiod of the interference signal.

The coherence microscope is in particular designed in such a way that ithas a depth variation which corresponds at least to the depth resolutionof the coherence microscope, determined by the coherence length of thelight emitted by the light source, and at most Nλ/4, wherein λ is thewavelength of the light emitted by the light source and N is the numberof sensor elements or the used sensor elements in the sensor line. Inthat case Nλ/4 represents the greatest depth variation for which thesensor line with N sensor elements or N sensor elements which are usedfulfils the scanning theorem when using light of the wavelength λ.

Typically the depth variation is in the region of about 100 μm but itcan also be below that and can be for example 20 μm or less. Inparticular it can also be in the region of the depth resolution of thecoherence microscope. The smaller the depth variation, thecorrespondingly shorter can be the sensor line used, and thus also therecording time for recording an image.

With the coherence microscope according to the invention, levels ofdepth resolution of 10 μm over a depth variation of about 100 μm arepossible without having to record a z-stack, and that considerablyshortens the scanning time for raster scanning of a specimen.

In an advantageous configuration the coherence microscope according tothe invention includes a point light source emitting measurement lightand at least one confocal aperture member. In that case the point lightsource can also be formed by the at least one confocal aperture member.In addition, there is a microscope optical system for focusing themeasurement light on to the specimen and for focusing the measurementlight reflected by the specimen on to the at least one confocal aperturemember which possibly at the same time forms the point light source, ora further confocal aperture member. The term confocal aperture member isintended in this respect to denote not only an aperture or slot disk butany confocally arranged optical element having an aperture or numericalaperture.

In the OCT in accordance with the state of the art stray light whichoriginates from others than the depths of the specimen region to beinvestigated has a disturbing effect on depth measurement. Such straylight is reduced in the coherence microscope according to the inventionby the confocal aperture member. Confocality serves in that respect notto increase lateral resolution but to permit measurement in sharplydelimited depth regions. It is therefore possible to operate with mediumor high apertures or numerical apertures (NA=between 0.1 and 0.5) whicheffectively block out the unwanted stray light but which nonethelessallow a high level of lateral resolution with which subcellularstructures can be recognised.

Preferably the aperture of the at least one confocal aperture member isso selected that the depth variation of the coherence microscopesubstantially corresponds to the depth extent of its confocal zone.

A further configuration of the coherence microscope has an optical fiberwhich feeds the measurement light to the microscope optical system. Inaddition preferably a scanning device is arranged between the opticalfiber and the microscope optical system. The at least one confocalaperture member can be formed in that case by the optical fiber. If amonomode fiber is used as the optical fiber, the optical distancecovered by the measurement light is established and known with a highlevel of accuracy.

In still a further configuration of the invention an ordered fiberbundle is connected between the optical fiber and the microscope opticalsystem, preferably between the scanning device and the microscopeoptical system. In that case the at least one confocal aperture membercan alternatively be formed by the optical fiber or by the fibers of thefiber bundle. As recording of z-stacks is not necessary with thecoherence microscope according to the invention, no mechanically movedelements are required at the distal end of the fiber. Equally there isno need to implement additional focusing devices at the distal end.

In a particular configuration of the invention the ordered fiber bundlecan be integrated into an endoscope. In that case the distal end of theendoscope can include a magnification optical system whose numericalaperture is so selected that the optical resolution at the fiber bundleend face corresponds to the diameter of the fibers of the fiber bundle.

A further configuration of the coherence microscope is distinguished inthat there is a scanning device for coupling measurement light into thefibers and/or for coupling measurement light reflected by the specimenout of the fibers. In a particular configuration provided between thescanning device and the proximal end of the ordered fiber bundle is anoptical system which is so designed that the light to be coupled intothe fibers is slightly defocused at the proximal end of the fiberbundle. It is possible in that way to ensure that each individual fiberis well affected in the same manner when the light is coupled in.Alternatively there can be a scanning control means which is adapted toperform an initialisation operation in which the central position of thefibers at the proximal end of the ordered fiber bundle is ascertained inorder to improve the coupling-in effect.

In an advantageous configuration of the invention the fibers of theordered fiber bundle are arranged linearly in mutually juxtaposedrelationship at the proximal end of the fiber bundle. That configurationpermits raster scanning with very high scanning frequencies. At the sametime it permits surface raster scanning with only one movable scanningelement. As the movable scanning element the scanning device can have inparticular a rotatable polygonal mirror.

The described operation of coupling the light in and out at the proximalend of the fiber bundle and/or the linear arrangement of the fibers atthe proximal end of the fiber bundle can be advantageously used not onlyin the coherence microscope according to the invention but also in otheritems of equipment in which light is to be coupled into and out ofoptical fiber bundles.

The numeral aperture and the magnification of the microscope opticalsystem of the coherence microscope according to the invention canadvantageously be so selected that lateral resolution approximatelycorresponds to the diameter of the fibers of the ordered fiber bundleand a maximum depth variation is reached.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features, properties and advantages of the present inventionwill be apparent to the man skilled in the art from the followingdetailed description of embodiments by way of example with reference tothe accompanying drawings in which:

FIG. 1 diagrammatically shows a first embodiment of the coherencemicroscope according to the invention,

FIG. 2 diagrammatically shows a second embodiment of the coherencemicroscope according to the invention, and

FIG. 3 diagrammatically shows light being coupled into and out of fibersof an optical fiber bundle.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Firstly the structure in principle of the coherence microscope accordingto the invention will be described with reference to FIG. 1. Themicroscope includes a light source 1 for emitting time-incoherent light,a divider 3 for dividing the light into a reference beam and ameasurement beam, a reference branch 5 into which the reference beam iscoupled from the divider 3 and in which it covers a defined distance, ameasurement branch 7 into which the measurement beam is coupled from thedivider and by way of which the measurement beam is supplied to thespecimen 13, and a detector 9 in which the measurement light reflectedby the specimen is superimposed with reference light from the referencebranch 5 and the superimposed light is detected.

The light source 1 is a wide band light source which emits substantiallytime-incoherent radiation. In the illustrated embodiment the lightsource 1 is a super-light emitting diode. Alternatively it is alsopossible to use other light sources if they emit light of a coherencelength which does not exceed a predetermined value such as for example alaser emitting short light pulses. The coherence length of the lightsource 1 determines the depth resolution of the optical coherencemicroscope. Besides the light source 1 the microscope includes a laserlight source 15, for example a laser diode, which emits radiation whichis coherent in respect of time, in the frequency range visible to thehuman eye. The laser light source 15 or the laser light emanatingtherefrom serves to be able to track the beam path of the light of thesuper-light emitting diode 1, which is emitted in the non-visible range.For that purpose, the laser light is mixed in a mixer 17 with the lightof the super-light emitting diode 1, the mixed beam being 90% from thesuper-light emitting diode 1 and 10% from the laser light source 15. Itwill be appreciated that other mixing ratios are also possible. Theradiation produced by the mixer 17 is coupled by the divider 3 90% intothe measurement branch and 10% into the reference branch. In thisrespect also other mixing ratios are possible.

The reference light beam is coupled into a reference light guide 6 inthe reference branch 5 and passed to a mirror 19 by way of an opticalsystem 18. The mirror 19 reflects the reference beam which afterreflection is coupled by the optical system 18 into the reference lightguide 6 again. A mixer 21 mixes the reflected reference light with thereference beam coming from the divider 3 in a 50:50 ratio and couplesthe light which is processed in that way into a further reference lightguide 23 which leads to the detector 9 and which passes the referencelight beam to a beam exit 25 of the reference branch 5. The referencelight guides are preferably monomode fibers.

The measurement light is passed by way of a measurement light guide 8arranged in the measurement branch 7 to a scanning device 32 by which itis directed on to a microscope optical system 28 for focusing themeasurement light beam on to a region of the specimen. The scanningdevice 32 includes a first galvanometer mirror 33 pivotable about anaxis for ascertaining an X-deflection of the measurement beam and asecond galvanometer mirror 35 pivotable about an axis for ascertaining aY-deflection of the measurement beam. The axes about which therespective galvanometer mirrors 33, 35 are pivotable are preferably inmutually perpendicular relationship but can also assume other anglesrelative to each other as long as they are not in mutually parallelrelationship. The galvanometer mirrors 33, 35 are controlled by means ofa scanning control means (not shown) in such a way that a lateralspecimen region is scanned step by step. In that case, in each scanningstep, the light reflected by the specimen 13 is received by themicroscope optical system 28 and fed to the measurement light guide 8again by way of the scanning device 32.

The numerical aperture NA of the measurement light guide 8 representsboth a point light source and also a confocal aperture member of thecoherence microscope. The numerical aperture and the magnification ofthe microscope optical system are advantageously so selected in thatrespect that the natural resolution of the microscope approximatelycorresponds to the diameter of a fiber (typically between 1 and 10 μm)and a maximum axial sharpness range is achieved. If the numericalaperture of a fiber is for example NA=0.18 an axial sharpness range of4λ/NA²=100 μm is reached and a lateral resolution of 0.5 μ/NA=2.2 isattained.

A mixer 27 to which the measurement light is passed by way of themeasurement light guide 8 mixes the measurement light reflected by thespecimen in a 50:50 ratio. The measurement light when processed in thatway is coupled by the mixer 27 into a further measurement light guide29, also preferably a monomode fiber, which passes the measurement lightto the beam exit 31 of the measurement branch 7.

From the beam exits 25, 31 of the reference branch 5 and the measurementbranch 7 respectively the reference light and the measurement light aredirected in the form of light cones 37, 39 on to a CCD line 41 as thesensor line of the detector 9, which represents the sensor surface ofthe detector 9. The two beam exits 25, 31 are arranged at a mutualspacing so that the two light cones are partially superimposed andsimultaneously illuminate at least a partial region 43 of the CCD line41. In the present embodiment the CCD line has 512 pixels which areessentially all irradiated by superimposed light. Interference phenomenaoccur only if the measurement light arriving at a point, that is to saya pixel, of the CCD line 41 has covered the same distance as thereference light arriving at the same point of the CCD line 41. A depthwithin the specimen 13 can be associated with the respective point onthe CCD line 41, from the known distances which the reference light hascovered from the beam exit 25 to the respective points on the CCD line.Only measurement light which has been reflected at that depth interfereswith the reference light at the associated point of the CCD line 41.

A read-out unit (not shown) reads out the CCD line and transmits theread-out data to an evaluation unit (also not shown) which effects theassociation of a pixel with the specimen depth from which themeasurement light impinging on the pixel originates. The operation ofreading out the CCD line can be effected with a high read-out rate byvirtue of the relatively low number of pixels which are to be read out.

In the described embodiment the high read-out rate can also be achievedif for example instead of a CCD line with 512 pixels, a CCD line with1024 or more pixels is used, of which only about 500 are irradiated withsuperimposed light and read out.

The recordings of the coherence microscope according to the inventionare distinguished in that a volume with an axial extent in the region ofthe depth of focus of the confocal microscope optical system is measuredat a given depth of the specimen. As the specimen is scanned in surfacemode in the lateral direction, very high data rates are involved. Theoverall system should therefore be designed and optimised for fast datadetection. The read-out rate of the CCD line which is necessary for thatpurpose is estimated hereinafter:

The depth variation Δz of the coherence microscope is given by:Δz=Nλ/2P,wherein N is the number of pixels of the CCD line, λ is the wavelengthof the light source (typically 800 mm) and P is the number of pixels perperiod of the interference signal. The number of pixels per periodshould be at least P=2 in order to comply with the scanning theorem. Ifthe arrangement is compared to a laser scan microscope, then the numberof recorded pixels in the lateral direction (X, Y) should typically be250×250=62500. With a 3D image frequency of 1 Hz the CCD lineconsequently must be read out at a line frequency of 62.5 kHz. Thelength of the CCD line, for a depth variation of Δz=100 μm and a periodof 2, is N=500. With a smaller depth variation the number of pixels perperiod can also be higher than 2; without a longer line having to beused. Alternatively however with a smaller depth variation the number ofpixels per period can also be maintained and in return the length of theCCD line can be reduced. As mentioned the number of pixels per periodshould be at least 2 and should advantageously be at most 4 in order toavoid unnecessarily long lines.

In the coherence microscope according to the invention, as in the caseof the OCT, depth resolution is determined by the coherence length ofthe light source 1. Each light source emits coherent light over a givenperiod of time, namely over the coherence time. In that respect lightsources with very short coherence times are viewed as beingtime-incoherent light sources. The coherence time can be converted intoa coherence length. Only those light beams whose distances covereddiffer by less than the coherence length can interfere with each other.The shorter the coherence length, the correspondingly more preciselymust the distances covered by the measurement beam and the referencebeam therefore coincide so that they can interfere with each other, thatis to say the difference in distances must be less than the coherencelength. In the case of a relatively short coherence length therefore itis possible to more accurately ascertain the depth region from which themeasurement beam was reflected, and that therefore permits better depthresolution for the microscope. Levels of depth resolution of 10 μm canbe achieved with the coherence lengths of common incoherent lightsources. With modern light sources it is possible to achieve levels ofresolution to below 1 μm. Preferably the depth resolution of thecoherence microscope is better than 20 μm, further preferably betterthan 10 μm and in particular better than 1 μm, in which respect depthresolution can depend on the desired use of the coherence microscope.

Further details of the structure of the detector, detection andascertainment of the depth profile from the levels of intensity detectedby the CCD line are described in DE 199 29 406, to the disclosure ofwhich reference is directed in this connection.

The volume ΔX, ΔY, ΔZ of the scattering specimen 13 can be measured witha high level of lateral (X, Y) and axial (Z) resolution with thecoherence microscope. The specimen 13 is raster scanned similarly to theconfocal laser scan microscope with the confocal microscope opticalsystem. In the coherence microscope according to the invention howeverthe confocality is not used as in the case of the confocal laser scanmicroscope to increase the degree of lateral resolution or to permitmeasurement in sharply delimited depth regions (<10 μm). Instead,confocality is used only to reduce extraneous light which originatesfrom outside the specimen region to be investigated.

In alternative configurations of the coherence microscope according tothe invention, in particular the scanning device 32, the galvanometermirrors 33, 35 can be replaced entirely or partially by other scanningelements such as for example rotatable polygonal mirrors.

A second embodiment of the coherence microscope according to theinvention is shown in FIG. 2. It differs from the first embodiment onlyin that disposed between the scanning device 32 and the microscopeoptical system 28 is a focusing lens 26 and an ordered fiber bundle 100which includes a number of optical fibers, preferably monomode fibers.

The measurement light is passed to the microscope optical system 28 byway of the fiber bundle 100. Introduction of the measurement light beaminto the proximal ends 106 of the optical fibers of the fiber bundle 100is effected by way of the focusing lens 26 with which the measurementlight beam is focused on to the entry faces of the fibers. The scanningdevice 32 which is designed as in the first embodiment makes it possiblein that respect for the measurement light beam to be directed on to thefocusing lens 26 in such a way that it is focused on to the proximal endof a selected fiber of the fiber bundle 100. The galvanometer mirrors33, 35 of the scanning device 32 are controlled by means of a scanningcontrol means (not shown) in such a way that the measurement light beamis successively introduced into all fibers or at least into a definedsubset of all fibers of the optical fiber bundle.

Coupling of the measurement light beam into the proximal ends of theindividual fibers of the ordered fiber bundle 100 can be improved in anumber of ways in order to achieve optimum coupling into the individualfibers with maximum scanning speed.

A possible way of improving coupling in the light, which is to beimplemented without major expenditure, provides that the light beamwhich is to be coupled into the fiber bundle 10 is not completelyfocused by means of the focusing lens 26 but is slightly defocused sothat, at the location of a fiber into which it is to be coupled, thearea of the defocused measurement light beam is somewhat larger than theentry faces of the fibers. It is possible in that way to ensure thateach individual fiber is well involved in the same manner. Defocusinghowever involves a signal loss which is not acceptable in all uses.

An alternative possible way of improving the coupling-in effect is tocontrol the scanning device 32 in such a way that each individual fiberof the fiber bundle 100 is optimally hit by the rastering light beam.The optimum setting for the scanning device is ascertained in aninitialisation step for each individual fiber. In the initialisationstep for example the proximal end 106 of the fiber bundle can be rasterscanned in a raster which is finer than that which arises out of thearrangement of the proximal ends of the individual fibers. Thereflections which occur upon raster scanning at the proximal end 106 ofthe fiber bundle 100 are stronger if they originate from an individualfiber than if they originate from the surrounding material in which theindividual fibers are embedded. It is therefore possible to ascertainthe exact position of the individual fibers by measurement of thereflections. Actuation by the scanning control means is then effected onthe basis of the positions ascertained in the initialisation step. Byvirtue of the fact that the reflections of the individual fibers arestronger than those of the surrounding material it is possible for thereflections also to be used for synchronisation of data recording.

Arranged at the distal end 102 of the fiber bundle is a confocalmicroscope optical system 28 with which the measurement light issuingfrom the fibers of the fiber bundle 100 is focused on to the specimen13. In addition, the microscope optical system 28 provides that thelight reflected by the specimen 13 is again focused on to the distal endof that fiber of the fiber bundle 100, from which it issued. The distalend of the fiber, that is to say its numerical aperture, represents inthat case both the point light source and also the confocal aperturemember of the confocal optical system. In this embodiment also it isadvantageous if the numeral aperture and the magnification of themicroscope optical system are so selected that the lateral resolution ofthe microscope approximately corresponds to the diameter of a fiber(typically 1 to 100 μm) and a maximum axial sharpness range is achieved.

Instead of being given by the numerical aperture of an individual fiberof the fiber bundle 100 the confocal aperture member, in the secondembodiment, can also be given by the numerical aperture of themeasurement light guide 8.

The measurement light reflected by the specimen 13 is passed by way ofthe fiber bundle 100 and the galvanometer mirrors 33, 35 of the scanningdevice 32 to a mixer 37 arranged in the measurement branch 7. The mixer27 mixes the measurement light reflected by the specimen 13 with thelight originating from the divider 3 in a 50:50 ratio. The measurementlight processed in that way is coupled into a measurement light guide29, preferably a monomode fiber, which passes the measurement light tothe beam exit 31 of the measurement branch 7. Superimposition of themeasurement light with the reference light and detection of thesuperimposed light are then effected as in the first embodiment.

An alternative embodiment of the scanning device 32 and the opticalfiber bundle 100 is now described with reference to FIG. 3. At itsdistal end 102 the optical fiber bundle 100 has the usual, almosthexagonal arrangement of the individual fibers 104. Unlike the usualfiber bundles the individual fibers 104 at the proximal end 106 of thefiber bundle 100 are however arranged in a line 105. If the fiber bundleincludes for example 50,000 individual fibers which are linearlyarranged at a spacing of 4 μm, the extent of the line 105 is 20 cm.

The scanning device 32 for scanning the fiber line 105 at the proximalend 106 of the fiber bundle 100 includes a rotatable polygonal mirror108 with a number of reflecting polygon surfaces 110, the axis ofrotation of which extends perpendicularly to the direction in which thefiber line extends. The measurement light beam is deflected by thereflecting polygon surfaces 110 in a direction towards the individualfibers 104 of the line 105. The arrangement of the polygonal mirror 108relative to the fiber line 105 is so selected that the line 105 israster scanned during a full revolution of the polygonal mirror 108 asoften as the polygonal mirror 108 has polygon surfaces 110. The lineconfiguration of the proximal end 106 of the fiber bundle 100, that isto say the linear arrangement of the individual fibers, thus permits anovel method of surface scanning in which deflection of the measurementlight beam for carrying out the surface scan is effected only in onedirection. Raster scanning of the line by means of the polygonal mirror108 permits very high scanning frequencies.

A complete depth profile is recorded with the coherence microscope atany point of the XY-plane (the so-called A-scan) without longitudinalscanning (z-scan) occurring. For specimens in which only a small depthregion is to be recorded, it is possible to use a short CCD line or along CCD line of which in each case only a short partial region is readout. The short line or the short partial region can be read out forperforming an A-scan at a high line frequency. In that way it ispossible to achieve very high measurement speeds when performing suchscans.

The coherence microscope according to the invention permits asimplification in the scanning procedure. Instead of a complete XY-scan,for example by means of an endoscope, the specimen is scanned along anXZ-plane, that is to say only along an X-line (the so-called B-scan).The X-direction can be adjusted both in its orientation and also in its‘width’ without fresh positioning of the optical system, for example ofthe endoscope, being necessary. That method permits the pathologist toperform a very fast optical biopsy which supplies a section in theaccustomed orientation. In particular the width of the one-dimensionalline can be adapted to the desired resolution and/or the desired signalstrength.

Essential areas of use of the coherence microscope according to theinvention are in optical biopsy and in in-vivo histology. The describedmethod is suitable for external uses (investigations on the skin and themucous membranes), for endoscopic diagnosis methods, in particular inthe gastrointestinal tract, and for ophthalmology investigations at theretina.

1. A coherence microscope including: a light source (1) emittingtime-incoherent light, a divider (3) for dividing the light emitted bythe light source (1) into measurement light which is supplied to aspecimen (13) and reflected thereby, and reference light; a point lightsource emitting the measurement light onto the specimen (13) and atleast one confocal aperture member; a microscope optical system (28) forfocusing the measurement light on the specimen (13) and for focusing themeasurement light reflected by the specimen on the at least one confocalaperture member, wherein the aperture of the at least one confocalaperture member is so selected that the depth extent of the confocalzone substantially corresponds to the depth stroke of the coherencemicroscope; a superimposition device (25, 31) for spatiallysuperimposing the measurement light reflected by the specimen (13) withthe reference light; and a sensor line (41) including a predeterminednumber of sensor elements for detecting the light resulting from thesuperimposition, the predetermined number of sensor elements beingselected so a read-out rate of at least 60 kHz is achieved; wherein thesuperimposition device has an emission device (25, 31) for emitting themeasurement light and the reference light which is adapted and arrangedrelative to the sensor line (41) such that extensive irradiation atleast of a part of the sensor line (41) with superimposed light iseffected and the ratio of the distances covered by the measurement lightand the reference light from the emission device (25, 31) to therespective impingement point on the sensor line (41) varies in theportion of the sensor line (41), that is irradiated with superimposedlight.
 2. A coherence microscope as set forth in claim 1 characterisedin that the sensor line (41) includes not more than about 1000 sensorelements.
 3. A coherence microscope as set forth in claim 1characterised in that the sensor line (41) includes not more than about500 sensor elements.
 4. A coherence microscope as set forth in claim 1characterised in that it has a depth variation which corresponds atleast to the depth resolution of the coherence microscope and at mostNλ/4, wherein λ is the wavelength of the light emitted by the lightsource (1) and N is the number of sensor elements in the sensor line(41).
 5. A coherence microscope as set forth in claim 4 characterised inthat its depth variation is 100 μm or less.
 6. A coherence microscope asset forth in claim 5 characterised in that its depth variation is 20 μmor less.
 7. A coherence microscope as set forth in claim 4 characterisedin that its depth variation substantially corresponds to its depthresolution.
 8. A coherence microscope as set forth in claim 1characterised in that there is an optical fiber (8) which feeds themeasurement light to the microscope optical system (28).
 9. A coherencemicroscope as set forth in claim 8 characterised in that the opticalfiber (8) is a monomode fiber.
 10. A coherence microscope as set forthin claim 8 characterised in that the at least one confocal aperturemember is formed by the optical fiber (8).
 11. A coherence microscope asset forth in claim 8 characterised in that an ordered fiber bundle (100)is interposed between the optical fiber (8) and the microscope opticalsystem.
 12. A coherence microscope as set forth in claim 11characterised in that the at least one confocal aperture member isformed by the optical fiber (8) or by the fibers (104) of the fiberbundle (100).
 13. A coherence microscope as set forth in claim 11characterised in that the ordered fiber bundle (100) is integrated intoan endoscope.
 14. A coherence microscope as set forth in claim 13characterised in that the microscope optical system (28) is arranged atthe distal end of the endoscope.
 15. A coherence microscope as set forthin claim 11 characterised in that the numerical aperture and themagnification of the microscope optical system (28) are so selected thatthe optical resolution at the fiber bundle end face corresponds to thediameter of the fibers (104) of the ordered fiber bundle (100).
 16. Acoherence microscope as set forth in claim 15 characterised in thatthere is provided a scanning device (32; 108) for coupling measurementlight into the fibers (104) of the ordered fiber bundle (100) and/or forcoupling measurement light reflected by the specimen (13) out of thefibers (104).
 17. A coherence microscope as set forth in claim 16characterised in that provided between the scanning device (32, 108) andthe proximal end (106) of the ordered fiber bundle (100) is an opticalsystem (26) which is so designed that it slightly defocuses the lightwhich is to be coupled into the fibers (104) at the proximal end (106)of the fiber bundle (100).
 18. A coherence microscope as set forth inclaim 16 characterised in that there is provided a scanning controlmeans which is adapted to perform an initialisation step in which thecentral position of the fibers (104) at the proximal end (106) of theordered fiber bundle is ascertained.
 19. A coherence microscope as setforth in claim 16 characterised in that the fibers (104) of the orderedfiber bundle (100) are arranged in linearly mutually juxtaposedrelationship at the proximal end (106) thereof.
 20. A coherencemicroscope as set forth in claim 19 characterised in that the scanningdevice (32) includes a rotatable polygonal mirror (108).
 21. A coherencemicroscope as set forth in claim 11 characterised in that the numericalaperture and the magnification of the microscope optical system (28) areso selected that the lateral resolution approximately corresponds to thediameter of the fibers (104) of the ordered fiber bundle (100) and amaximum depth variation is achieved.
 22. A coherence microscope as setforth in claim 1 characterised in that the sensor line (41) includes atleast 1000 sensor elements and wherein not more than about 1000 sensorelements are used.
 23. A coherence microscope as set forth in claim 1characterised in that the sensor line (41) includes at least 500 sensorelements and wherein not more than about 500 sensor elements are used.24. A coherence microscope comprising: a light source (1) emittingtime-incoherent light, a divider (3) for dividing the light emitted bythe light source (1) into measurement light which is supplied to aspecimen (13) and reflected thereby, and reference light; asuperimposition device (25, 31) for spatially superimposing themeasurement light reflected by the specimen (13) with the referencelight; a short sensor line (41) or a long sensor line only a portion ofthe sensor elements of which are used for detecting the light resultingfrom the superimposition, the sensor line being so short or only such aportion of sensor elements is used that a read-out rate of at least 60kHz is achieved; an emission device (25, 31) for emitting themeasurement light and the reference light from the superimpositiondevice, the emission device being adapted and arranged relative to thesensor line (41) such that extensive irradiation at least of a part ofthe sensor line (41) with superimposed light is effected and the ratioof the distances covered by the measurement light and the referencelight from the emission device (25, 31) to the respective impingementpoint on the sensor line (41) varies in the portion of the sensor line(41), that is irradiated with superimposed light; a point light sourceemitting the measurement light onto the specimen (13) and at least oneconfocal aperture member; a microscope optical system (28) for focusingthe measurement light onto the specimen (13) and for focusing themeasurement light reflected by the specimen on the at least one confocalaperture member; an optical fiber (8) which feeds the measurement lightto the microscope optical system (28) and an ordered fiber bundle (100)which is interposed between the optical fiber (8) and the microscopeoptical system (28), the fibers (104) of the ordered fiber bundle (100)being arranged in linearly mutually juxtaposed relationship at theproximal end (106) thereof; and a scanning device including a rotatablepolygonal mirror (108) for coupling measurement light into the fibers(104) of the ordered fiber bundle (100) and/or for coupling measurementlight reflected by the specimen (13) out of the fibers (104).